POJ 3(2):52-56(2010) ISSN:1836-3644 Induction of protonemal gemmae and gametophyte of Cratoneuron decipien (Brid.) G. Roth using IAA and kinetin Md. Giush Uddin Ahmed and Cheol Hee Lee* Department of Horticultural Sciences, Chungbuk National University, Cheongju 361-763, Korea *Corresponding author: [email protected] Abstract The protonemal gemmae and gametophyte inducing effect of the IAA and kinetin was examined in moss Cratoneuron decipiens growing using solid media. Cultures were raised from gametophyte tips on a solid basal medium containing Knop’s major salts and Nitsch and Nitsch’s trace elements. Aseptic chopped gametophytes were inoculated onto four types of concentrations (10-8, 10-7, 10-6 and 10-5 M) of IAA and kinetin. High frequency of bud and gemmae were selected then subcultured to same supplemented of IAA and kinetin. Protonema produced gemmae, callus and gametophytic bud. Production of protonemal gemmae varies according to the concentration of IAA and kinetin, whereas low concentrations of growth regulators promoted gemmae formation and bud induction. Gametophytes were regenerated from protonema and calli after subculture. IAA regulated gametophytes induction and growth. Kinetin influenced gemmae formation and gametophyte regeneration also. All aspects of development of this moss species are governed by the external growth regulators. Keywords: Gemmae, gametophyte, growth regulators, protonema. Abbreviations: IAA, indole-3-acetic acid; M, mole; NaOCl, sodium hypochlorite; ANOVA, analysis of variance. Introduction only by the internal environment, but also by the external environment. These responses are typically mediated by Mosses are clonal organisms. All mosses are able to the hormones. Known bryophytes hormones regulate spread vegetatively through fragments and propagules. growth and gametangial production, protonemal bud Mosses vegetative reproduction includes fragments, formation, branching gemmae formation and senescence. gemmae and vegetative diasporas. Magill (1990) like- The mode of control of these growth regulators are wise used the concept of Goebel to define gemmae as poorly understood in bryophytes, although in most cases vegetative diaspores with no apical cell and that always they seem to act similarly to their mode of action in must begin growth with a protonemal phase. These units tracheo- phytes. The effect of IAA and kinetin on the then include caducous leaves and endogenous gemmae, gemmae formation and gametophyte induction is as well as those specialized, oval, round, or irregularly reported here. shaped structures we have always called gemmae in the strictest sense. In vitro, C. decipiens produces gemmae as Materials and methods well as gametophytic buds on protonema. Mosses hormones operate very much as they do in Plant materials and culture establishment tracheophytes (Maravolo, 1980). In bryophytes, auxins are transported directionally, permitting apical domina- Cratoneuron decipien (Brid.) G. Roth was collected from nce to occur, and their activity is concentration Cheongju, S. Korea. Mature gametophytes were main- dependent. The highest concentrations of auxin occur at tained in 50% shady places under greenhouse condition. the tip and base of the upright gametophore, with Mature gametophytes tip were surface sterilized with 1% distribution throughout the stem, as demonstrated in NaOCl water for 1 minute and washed repeatedly with Physcomitrella patens (Bierfreund et al., 2003). sterile water. Gametophyte tips were cultured on a solid Cytokinins can increase gametophyte production in basal medium containing Knop’s (1865) major salts and several mosses (Bopp, 1963). Hahn and bopp (1968) Nitsch and Nitsch’s (1956) trace elements solution with have employed budding as a specific test for exogenous 1% sucrose, 0.8% agar and pH was adjusted to 5.7. The cytokinins. Developments of mosses are influenced not cultures were maintained under an illumination of 2000±200 lx for 14h/d, at 25±1 ºC. 52 Table 1. Effect of IAA and kinetin on differentiation of Cratoneuron decipiens protonema. IAA Kinetin 10-8M 10-7M 10-6M 10-5M 10-8M 10-7M 10-6M 10-5M Number of bud/culture 31.4±1.2 27.4±1.5 17.1±0.5 5.3±0.9 46.7±2.3 33.5±1.8 25.2±1.1 17.3±1.5 vessel (mean±SE) Size of budz +++ ++ + + +++ +++ ++ + Estimated 20-30% 20-30% 10-20% 5-10% 30-40% 20-30% 20-30% 10-20% caulonema z -, no bud development; +, small buds, most of them few cells; ++, buds with more than 10 cells, +++, large buds, some leaves developed A1 A2 A3 A4 A1 A2 A3 A4 B1 B2 B3 B4 B1 B2 B3 B4 -8 Fig 1. Effect of IAA (A1-4) and Kinetin (B1-4) on gemmae induction from secondary protonema. A1= 10 M of IAA, -7 -6 protonemal branches showing round shaped gemmae; A2= 10 M of IAA, developing composite gemmae; A3= 10 M of -5 -8 IAA, composite gemmae; A4= 10 M of IAA, mature, composite gemmae. B1= 10 M kinetin and protonema branches with -7 -6 early stage of gemmae; B2= 10 M kinetin and formation of composite gemmae; B3= 10 M of kinetin and composite -5 gemmae ready for germination; B4= 10 M kinetin, composite gemmae germinated to protonema. Photographs were taken from 4 weeks of subcultures. Gemmae induction Experimental setup and statistical analysis Aseptic chopped gametophyte (1g inoculums per 300ml All experiments were repeated 3 times. Parameters was culture vessel) were inoculated onto four types of analyzed by one-way ANOVA with DMRT (Duncan concentrations (10-8, 10-7, 10-6 and 10-5 M) of IAA and multiple range test) post hoc test. All analysis was carried kinetin under same cultivation conditions. After two out using PASW Statistics 17. Photographs of gemmae weeks, caulonemata were observed using stereomicro- cells were taken using AM423X-Dino-Eye USB digital scope. Then three weeks of incubation, 1g of proto- eyepiece camera. nemata was taken from the surface of solid medium and gemmae and buds were observed. A high frequency of Results bud and gemmae was chosen for next subculture. Culture establishment Gametophyte induction Protonemata were developed from inoculated gameto- After 30 days of culture, High frequency of bud and phyte tips. One month of inoculation gametophyte tips gemmae were selected and subcultured to four types of produced many erect gametophytes. About 5-10 gamet- supplemented of IAA and kinetin (10-8, 10-7, 10-6 and 10-5 ophytes formed around each gametophyte tip. Secondary M) under same cultivation conditions. protonema also developed from inoculated gametophyte tips. 53 Table 2. Effect of IAA for in vitro gametophyte production of Cratoneuron decipiens moss. Con. of IAA Gametophyte No. of Fresh weight Dry weight (mg)/flax (M) height (cm) gametophyte/flax (mg)/flax 10-8 1.36±0.10 a 388.20±11.12 a 874.56±2.77 a 179.26±0.81 a 10-7 1.00±0.05 b 322.20±6.38 b 770.46±5.35 b 156.38±1.42 b 10-6 0.56±0.05 c 272.80±6.10 c 646.62±10.29 c 126.21±1.68 c 10-5 0.32±0.37 d 194.45±5.87 d 366.22±9.90 d 88.83±2.58 d Data from 60 days old culture and represents mean with standard errors and data followed by the same letter are not significantly different at 5% level (DMRT test). Table 3. Effect of Kinetin for in vitro gametophyte production of Cratoneuron decipiens moss. Con. of Gametophyte No. of Fresh weight Dry weight (mg)/flax Kinetin (M) height (cm) gametophyte/flax (mg)/flax 10-8 2.90±0.11 a 225.40±6.81 a 2738.20±31.48 a 358.10±4.19 a 10-7 2.54±0.14 a 125.20±7.59 b 1602.00±59.29 b 219.18±8.33 b 10-6 2.02±0.05 b 99.80±3.18 c 1163.80±6.76 c 215.38±1.14 b 10-5 0.86±0.15 c 77.60±2.89 d 1134.80±7.63 c 176.34±1.60 c Data from 60 days old culture and represents mean with standard errors and data followed by the same letter are not significantly different at 5% level (DMRT test). Proliferation of C. decipiens was successfully give rise gemmae germinating on the same medium (i.e. medium enough, purified and sterile plant materials. on which they were produced) developed two or three filaments of restricted growth (Figure 1B3). This in turn Gemmae induction formed gemmae at their tips, thus resulting in composite gemmae (Figure 1 A3, A4, B3 B4). After subculture of Fine chopped gametophytes were tested to produce gemmae to higher concentration of growth regulators, secondary protonema and then induced callus, gemmae produced normal protonema (Figure 1 B4). The colors of and gametophytic bud. Chopped gametophytes on solid gemmae were light to brown with the increase of growth medium were allowed to grow numerous gametophytes regulators. through secondary protonema and fragmentation. Table 1 summarizes the effects of exogenous IAA and kinetin on Gametophyte induction differentiation of protonema. The media containing 10-8, 10-7, 10-6, 10-5M IAA induced 31.4±1.2, 27.4±1.5, The medium with 10-8M of kinetin was produced highest 17.1±0.5 and 5.3±0.9 buds per culture vessel frequency of buds and gemmae and it was selected for respectively. Kinetin induced higher number of bud than source of gametophyte induction. Biomass (calli, IAA and it was 46.7±2.3, 33.5±1.8, 25.2±1.1 and protonema, gemmae and buds) from selected lines were 17.3±1.5 buds per culture vessel with the media subcultured onto 10-8, 10-7, 10-6, 10-5M of IAA and containing 10-8, 10-7, 10-6, 10-5M kinetin respectively.